This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic 06-DE-102 Structural and Molecular Atlases of Craniofacial Development. We propose to construct a developmental atlas for the craniofacial skeleton of zebrafish, a widely used model organism for understanding conserved features of development of the skull, and human disorders such as cleft palate. The atlas will provide a key resource for scientists wishing to discover how the complex anatomy of the zebrafish craniofacial skeleton develops. It will serve as a critical reference for learning the defects of the many zebrafish craniofacial mutants now available for study, as well as new mutants now being generated through genome-wide initiatives. It also will be useful for understanding development of the zebrafish skull compared with other organisms, such as mouse. The atlas will be web-based and freely available on FaceBase, a web resource rapidly becoming the standard for understanding craniofacial development, and on ZFIN, the specific informatics resource for zebrafish development and genetics. It will include a series of developmental stages beginning with the 1-day postfertilization embryo when the skeletal primordia are present, but bones and cartilages have not yet formed. It will continue with selected stages through the 3-week-old juvenile stage when nearly all of the elements of the skull are present, and shaped approximately as in the adult, but of miniature size. It will be 3-dimensional and interactive: The user will be able to virtually dissect the skull - calling up particular subsets of elements of interest, to rotate the image and observe a set of elements from any point of view, to section through the structure in any plane, and to examine how the structure changes at different developmental stages. A key innovation is that the atlas will be based entirely on images taken of the living, developing organism expressing transgenic fluorescent proteins. These markers will be driven by the promoters of key skeletal regulatory genes, including fli1, expressed in all craniofacial mesenchyme, and dlx5a, expressed in a subset of this mesenchyme that is restricted to the ventral elements of the skull, including the lower jaw. During stages of skeletal morphogenesis and growth, the cells making the skeleton will be revealed specifically by expression of markers including sox10 and foxp2a for cartilage, and osterix for bone. The fish expressing one of these markers also will be vitally counterstained with a fluorescent dye such as Alizarin red to reveal the mineralized bone. The atlas will feature, for every stage, high magnification, through-focus, 2-color image stacks made by confocal microscopy. Such imaging takes full advantage of the many currently available transgenic constructs, the optical clarity of the zebrafish, and of its relatively small size and the relatively small number of cells that build the skull. These attributes combine to provide cell-level resolution with unsurpassed specificity and detail throughout an extended period of development. The atlas will also include, especially for the later developmental stages, medium-resolution images at lower magnification made by optical projection tomography (OPT). The user will be able to interactively manipulate these OPT stacks in the same way as for the confocal stacks, and will allow better 3-d understanding of the more global features of skull organization and development. PUBLIC HEALTH RELEVANCE: The zebrafish craniofacial atlas that will come from this project will provide a resource for skeletal anatomy in an animal model that is valuable for understanding human craniofacial disorders such as cleft palate. The resource will provide for understanding normal morphological developmental in zebrafish as well as being a reference for diagnosing the nature of craniofacial mutant phenotypes.